Multiplexed Surface Plasmon Resonance Imaging for Protein Biomarker Analysis

  • Eric Ouellet
  • Louise Lund
  • Eric T. LagallyEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 949)


The reliable detection of ligand and analyte binding is of significant importance for the field of medical diagnostics. Recent advances in proteomics and the rapid expansion in the number of identified protein biomarkers enhance the need for reliable techniques for their identification in complex samples. Surface plasmon resonance imaging (SPRi) provides label-free detection of this binding process in real-time. This chapter details the fabrication of an SPR imaging instrument and its use in analyzing molecular binding interactions with the use of a high-density microfluidic SPRi chip, capable of multiplexed analysis as well as various immobilization chemistries. Controlled recovery of bound biomarkers is demonstrated to enable their identification using mass spectrometry. Finally, activated leukocyte cell adhesion molecule (ALCAM), a protein biomarker associated with a variety of cancers, is identified from human crude cell lysates using the microfluidic surface plasmon resonance imaging (SPRi) instrument.

Key words

Surface plasmon resonance (SPR) imaging Microfluidic arrays Poly(dimethylsiloxane) PDMS HeLa cells Biomarkers Mass spectrometry Self-assembled monolayers 



The authors would like to thank Dr. Leonard Foster for his advice and continued support, Dr. Leroy Hood and Christopher Lausted for their contributions to the microfluidic device design, Anders Riss Kristensen for his technical assistance with HeLa cell culture and Dr. Robert Parker for technical assistance with mass spectrometry analysis. Microfabrication was performed in the UBC Nanolab.


  1. 1.
    Piliarik M, Vaisocherová H, Homola J (2005) A new surface plasmon resonance sensor for high-throughput screening applications. Biosens Bioelectron 20:2104–2110CrossRefGoogle Scholar
  2. 2.
    Wassaf D, Kuang G, Kopacz K, Wu Q-L, Nguyen Q, Toews M, Cosic J, Jacques J, Wiltshire S, Lambert J, Pazmany CC, Hogan S, Ladner RC, Nixon AE, Sexton DJ (2006) High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays. Anal Biochem 351:241–253CrossRefGoogle Scholar
  3. 3.
    Shumaker-Parry JS, Aebersold R, Campbell CT (2004) Parallel, quantitative measurement of protein binding to a 120-element double-stranded DNA array in real time using surface plasmon resonance microscopy. Anal Chem 76:2071–2082CrossRefGoogle Scholar
  4. 4.
    Boozer C, Kim G, Cong S, Guan H, Londergan T (2006) Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review of new surface plasmon resonance technologies. Curr Opin Biotechnol 17:400–405CrossRefGoogle Scholar
  5. 5.
    Luo Y, Yu F, Zare RN (2008) Microfluidic device for immunoassays based on surface plasmon resonance imaging. Lab Chip 8:694–700CrossRefGoogle Scholar
  6. 6.
    Rothenhausler B, Knoll W (1988) Surface–plasmon microscopy. Nature 332:615–617CrossRefGoogle Scholar
  7. 7.
    Yeatman E, Ash EA (1987) Surface plasmon microscopy. Electron Lett 23:1091–1092CrossRefGoogle Scholar
  8. 8.
    Campbell CT, Kim G (2007) SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics. Biomaterials 28:2380–2392CrossRefGoogle Scholar
  9. 9.
    Homola J, Yee SS, Gauglitz G (1999) Surface plasmon resonance sensors: review. Sens Actuators B Chem 54:3–15CrossRefGoogle Scholar
  10. 10.
    Shumaker-Parry JS, Zareie MH, Aebersold R, Campbell CT (2004) Microspotting streptavidin and double-stranded DNA arrays on gold for high-throughput studies of protein-DNA interactions by surface plasmon resonance microscopy. Anal Chem 76:918–929CrossRefGoogle Scholar
  11. 11.
    Li Y, Wark AW, Lee HJ, Corn RM (2006) Single-nucleotide polymorphism genotyping by nanoparticle-enhanced surface plasmon resonance imaging measurements of surface ligation reactions. Anal Chem 78:3158–3164CrossRefGoogle Scholar
  12. 12.
    Malic L, Veres T, Tabrizian M (2009) Biochip functionalization using electrowetting-on-dielectric digital microfluidics for surface plasmon resonance imaging detection of DNA hybridization. Biosens Bioelectron 24:2218–2224CrossRefGoogle Scholar
  13. 13.
    Goodrich TT, Lee HJ, Corn RM (2004) Direct detection of genomic DNA by enzymatically amplified SPR imaging measurements of RNA microarrays. J Am Chem Soc 126:4086–4087CrossRefGoogle Scholar
  14. 14.
    Wegner GJ, Lee HJ, Marriott G, Corn RM (2003) Fabrication of histidine-tagged fusion protein arrays for surface plasmon resonance imaging studies of protein–protein and protein–DNA interactions. Anal Chem 75:4740–4746CrossRefGoogle Scholar
  15. 15.
    Li Y, Lee HJ, Corn RM (2007) Detection of protein biomarkers using RNA aptamer microarrays and enzymatically amplified surface plasmon resonance imaging. Anal Chem 79:1082–1088CrossRefGoogle Scholar
  16. 16.
    Wegner GJ, Lee HJ, Corn RM (2002) Characterization and optimization of peptide arrays for the study of epitope–antibody interactions using surface plasmon resonance imaging. Anal Chem 74:5161–5168CrossRefGoogle Scholar
  17. 17.
    Lee K-H, Su Y-D, Chen S-J, Tseng F-G, Lee G-B (2007) Microfluidic systems integrated with two-dimensional surface plasmon resonance phase imaging systems for microarray immunoassay. Biosens Bioelectron 23:466–472CrossRefGoogle Scholar
  18. 18.
    Lausted CG, Hu Z, Hood LE (2008) Quantitative serum proteomics from surface plasmon resonance imaging. Mol Cell Proteomics 7(12):2464–2474CrossRefGoogle Scholar
  19. 19.
    Fu E, Chinowsky T, Nelson K, Johnston K, Edwards T, Helton K, Grow M, Miller JW, Yager P (2007) SPR imaging-based salivary diagnostics system for the detection of small molecule analytes. Ann N Y Acad Sci 1098:335–344CrossRefGoogle Scholar
  20. 20.
    Rich RL, Myszka DG (2007) Higher-throughput, label-free, real-time molecular interaction analysis. Anal Biochem 361:1–6CrossRefGoogle Scholar
  21. 21.
    Bravman T, Bronner V, Lavie K, Notcovich A, Papalia GA, Myszka DG (2006) Exploring “one-shot” kinetics and small molecule analysis using the ProteOn XPR36 array biosensor. Anal Biochem 358:281–288CrossRefGoogle Scholar
  22. 22.
    Stroock AD, Dertinger SKW, Ajdari A, Mezic I, Stone HA, Whitesides GM (2002) Chaotic mixer for microchannels. Science 295:647–651CrossRefGoogle Scholar
  23. 23.
    Rich RL, Cannon MJ, Jenkins J, Pandian P, Sundaram S, Magyar R, Brockman J, Lambert J, Myszka DG (2008) Extracting kinetic rate constants from surface plasmon resonance array systems. Anal Biochem 373:112–120CrossRefGoogle Scholar
  24. 24.
    Barry R, Ivanov D (2004) Microfluidics in biotechnology. J Nanobiotechnol 2:2CrossRefGoogle Scholar
  25. 25.
    Bange A, Halsall HB, Heineman WR (2005) Microfluidic immunosensor systems. Biosens Bioelectron 20:2488–2503CrossRefGoogle Scholar
  26. 26.
    deMello AJ (2006) Control and detection of chemical reactions in microfluidic systems. Nature 442:394–402CrossRefGoogle Scholar
  27. 27.
    Khandurina J, Guttman A (2002) Bioanalysis in microfluidic devices. J Chromatogr A 943:159–183CrossRefGoogle Scholar
  28. 28.
    Wang Z, Wilkop T, Xu D, Dong Y, Ma G, Cheng Q (2007) Surface plasmon resonance imaging for affinity analysis of aptamer–protein interactions with PDMS microfluidic chips. Anal Bioanal Chem 389:819–825CrossRefGoogle Scholar
  29. 29.
    Ouellet E, Lausted C, Lin T, Yang CWT, Hood L, Lagally ET (2010) Parallel microfluidic surface plasmon resonance imaging arrays. Lab Chip 10:581–588CrossRefGoogle Scholar
  30. 30.
    Burkhardt M, Mayordomo E, Winzer K-J, Fritzsche F, Gansukh T, Pahl S, Weichert W, Denkert C, Guski H, Dietel M, Kristiansen G (2006) Cytoplasmic overexpression of ALCAM is prognostic of disease progression in breast cancer. J Clin Pathol 59:403–409CrossRefGoogle Scholar
  31. 31.
    Vaisocherová H, Faca VM, Taylor AD, Hanash S, Jiang S (2009) Comparative study of SPR and ELISA methods based on analysis of CD166/ALCAM levels in cancer and control human sera. Biosens Bioelectron 24:2143–2148CrossRefGoogle Scholar
  32. 32.
    Kristiansen G, Pilarsky C, Wissmann C, Stephan C, Weissbach L, Loy V, Loening S, Dietel M, Rosenthal A (2003) ALCAM/CD166 is up-regulated in low-grade prostate cancer and progressively lost in high-grade lesions. Prostate 54:34–43CrossRefGoogle Scholar
  33. 33.
    Faca VM, Song KS, Wang H, Zhang Q, Krasnoselsky AL, Newcomb LF, Plentz RR, Gurumurthy S, Redston MS, Pitteri SJ, Pereira-Faca SR, Ireton RC, Katayama H, Glukhova V, Phanstiel D, Brenner DE, Anderson MA, Misek D, Scholler N, Urban ND, Barnett MJ, Edelstein C, Goodman GE, Thornquist MD, McIntosh MW, DePinho RA, Bardeesy N, Hanash SM (2008) A mouse to human search for plasma proteome changes associated with pancreatic tumor development. PLoS Med 5:e123CrossRefGoogle Scholar
  34. 34.
    Pitteri SJ, JeBailey L, Faça VM, Thorpe JD, Silva MA, Ireton RC, Horton MB, Wang H, Pruitt LC, Zhang Q, Cheng KH, Urban N, Hanash SM, Dinulescu DM (2009) Integrated proteomic analysis of human cancer cells and plasma from tumor bearing mice for ovarian cancer biomarker discovery. PLoS One 4:e7916CrossRefGoogle Scholar
  35. 35.
    Rosso O, Piazza T, Bongarzone I, Rossello A, Mezzanzanica D, Canevari S, Orengo AM, Puppo A, Ferrini S, Fabbi M (2007) The ALCAM shedding by the metalloprotease ADAM17/TACE is involved in motility of ovarian carcinoma cells. Mol Cancer Res 5:1246–1253CrossRefGoogle Scholar
  36. 36.
    van Kempen LCLT, van den Oord JJ, van Muijen GNP, Weidle UH, Bloemers HPJ, Swart GWM (2000) Activated leukocyte cell adhesion molecule/CD166, a marker of tumor progression in primary malignant melanoma of the skin. Am J Pathol 156:769–774CrossRefGoogle Scholar
  37. 37.
    Rappsilber J, Ishihama Y, Mann M (2002) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75:663–670CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media,LLC 2013

Authors and Affiliations

  • Eric Ouellet
    • 1
    • 2
    • 3
  • Louise Lund
    • 1
    • 4
  • Eric T. Lagally
    • 1
    • 2
    Email author
  1. 1.Michael Smith LaboratoriesUniversity of British ColumbiaVancouverCanada
  2. 2.Department of Chemical and Biological EngineeringUniversity of British ColumbiaVancouverCanada
  3. 3.Biomedical Engineering ProgramUniversity of British ColumbiaVancouverCanada
  4. 4.Center for High Throughput BiologyUniversity of British ColumbiaVancouverCanada

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